The present invention relates to novel hybrid plasmid vectors, recombinant plasmids containing a nitrile hydratase gene and an amidase gene, transformants that are made by transforming bacteria belonging to the genus Rhodococcus or Escherichia with the recombinant plasmids and to methods of producing amides and acids.
Microorganisms belonging to the genus Rhodococcus are known to hydrate or hydrolyze nitriles to produce amides or acids (EP Publication No. 0188316, 0204555, and 0348901). In addition, microorganisms belonging to Rhodococcus rhodochrous are known to have a high hydration activity of nitriles (EP Publication No. 0307926). These useful properties of the microorganisms have motivated to develop a host-vector system to utilize bacteria belonging to the genus Rhodococcus. In reality, vectors suitable to host bacteria belonging to the genus Rhodococcus have not been appreciably developed yet. Very few suitable plasmid vectors have been found from Rhodococcus sp. H13-A [J.Bacteriol. 170:638-645 (1988)], Rhodococcus erythropolis (rhodochrous) ATCC12674 [Mol.Gen. Genet., 211:148-154 (1988)] and Rhodococcus rhodochrous ATCC 4276 which was disclosed by the present inventors in Japanese Patent Application No. 270377/1990. The development of novel vectors which are derived from the genus Rhodococcus and industrially useful in culturing microorganisms or in improving the properties of microorganisms, has been awaited.
The present inventors have isolated genes encoding nitrile degrading enzymes from bacteria belonging to the genus Rhodococcus, cloned the genes into vectors derived from Escherichia coli and investigated the gene expression in Escherichia coli hosts. The enzymes having a sufficient enzymatic activity have not been produced in Escherichia coli hosts so far [Eur.J.Bichem. 181:563-570 (1989), Biochem.Biophys.Acta., 1088: 225-233 (1991)].
Industrially useful, covalently closed circular plasmids derived from bacteria belonging to the genus Rhodococcus are potentially suitable plasmids to genetically improve the desirable properties of the genus Rhodococcus hosts. These plasmids, however, do not contain drug resistance genes to be used as a marker. Suitable plasmid vectors could be constructed by introducing a marker gene into the plasmids. There is a single example that is described in J. Bacteriol. 170: 638-645, 1988.
We have successfully constructed shuttle vectors suitable for industrial use by inserting drug resistance genes to be used as a marker, cloning sites and genes necessary for replication in Escherichia coli into covalently closed circular plasmids such as pRC001, pRC002. pRC003 and pRC004. We have subsequently subcloned the gene of a clone encoding a nitrile degradation enzyme into the shuttle vectors to give recombinant plasmids, transformed bacteria belonging to the genus Rhodococcus or Escherichia with the recombinant plasmids and produced amides and acids using the transformants.